In vitro characterization of baloxavir acid, a first-in-class cap-dependent endonuclease inhibitor of the influenza virus polymerase PA subunit.

Cap-dependent endonuclease (CEN) resides in the PA subunit of the influenza virus and mediates the critical "cap-snatching" step of viral RNA transcription, which is considered to be a promising anti-influenza target. Here, we describe in vitro characterization of a novel CEN inhibitor, baloxavir acid (BXA), the active form of baloxavir marboxil (BXM). BXA inhibits viral RNA transcription via selective inhibition of CEN activity in enzymatic assays, and inhibits viral replication in infected cells without cytotoxicity in cytopathic effect assays. The antiviral activity of BXA is also confirmed in yield reduction assays with seasonal type A and B viruses, including neuraminidase inhibitor-resistant strains. Furthermore, BXA shows broad potency against various subtypes of influenza A viruses (H1N2, H5N1, H5N2, H5N6, H7N9 and H9N2). Additionally, serial passages of the viruses in the presence of BXA result in isolation of PA/I38T variants with reduced BXA susceptibility. Phenotypic and genotypic analyses with reverse genetics demonstrate the mechanism of BXA action via CEN inhibition in infected cells. These results reveal the in vitro characteristics of BXA and support clinical use of BXM to treat influenza.

Effects of methyl-beta-cyclodextrin treatment on secretion profile of interferon-beta and zonula occuludin-1 architecture in Madin-Darby canine kidney cell monolayers.

The interferon (IFN) is a paradigm of secretory protein. However, it has been poorly understood how its secretion is regulated in polarized epithelial cells. Recently, we had shown that exogenous IFNs transiently expressed in polarized monolayers were predominantly secreted to the side on which gene transfection had been performed, while stably expressed IFNs were secreted almost equally to the both cell sides. Since those modes of secretion did not affect each other, epithelial cell layers seemed to have at least two protein sorting/secretion pathways, one for transient expression and the other for stable expression, for identical secretory proteins. Furthermore, this dual secretion profile seemed to be mediated by distinct post-trans Golgi network vesicles, suggesting the involvement of lipid rafts in the sorting multiplicity. To address this issue, here we studied the effects of cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD) on the secretion profile of IFN-beta exogenously expressed in Madin-Darby canine kidney (MDCK) cells. The MbetaCD-treatment, however, did not affect the profile in either transient or stable expression, although the architecture of zonula occuludin-1, which links to the tight junction, was substantially disrupted by the treatment. Further analysis of Triton X-100-insoluble cell extracts by sucrose density centrifugation demonstrated that IFN-beta was not apparently associated with lipid rafts in either transient or stable expression. These results suggest that lipid rafts may not be crucially involved in the regulation of secretion polarity of IFN-beta in the epithelial cells.

Cholesterol is required for the polarized secretion of erythropoietin in Madin-Darby canine kidney cells.

It has already been reported that stably expressed exogenous human wild-type EPO (wtEPO) is preferentially secreted to the apical side and one of the three N-linked carbohydrate chains critically acts as an apical sorting determinant in Madin-Darby canine kidney (MDCK) cells. It has been suggested that lipid rafts are involved in the apical sorting of membrane and secretory proteins. To investigate the involvement of lipid rafts in the apical sorting of wtEPO, we examined the effect of cholesterol depletion with methyl-beta-cyclodextrin on the secretion polarity of EPO and analyzed Triton X-100 insoluble cell extracts by sucrose density gradients centrifugation in MDCK cells. We found that wtEPO was shifted in non-polarized direction by cholesterol depletion. Most of the wtEPO was not detectable in the raft fractions by sucrose density gradients centrifugation analysis. These results indicate that apical secretion of EPO involves a cholesterol-dependent mechanism probably not involving lipid rafts.

Simultaneous detection of DsRed2-tagged and EGFP-tagged human beta-interferons in the same single cells.

The red fluorescent protein DsRed2 is a useful fusion tag for various proteins, together with the enhanced green fluorescent protein (EGFP). These chromoproteins have spectral properties that allow simultaneous distinctive detection of tagged proteins in the same single cells by dual color imaging. We used them for tagging a secretory protein, human interferon-beta (IFN-beta). Expression plasmids for human IFN-beta tagged with DsRed2 or with EGFP at the carboxyl terminal were constructed and their coexpression was examined in Mardin-Darby canine kidney epithelial cells. Although maturation of DsRed2 for coloration was slow and the color intensity was weak compared with EGFP, low temperature treatment (20 degrees C) allowed DsRed2-tagged human IFN-beta to be detected in the cells using color imaging. Consequently, the two chimeric proteins were shown to be colocalized in the same single cells by dual color confocal microscopy. This approach will be useful for investigating subcellular localization of not only cell resident proteins but also secretory proteins.